All of brain function, from sensory perception to behavior, is derived from the pattern and properties of the synaptic connections among billions (in humans) of individual neurons. The long-term goal of this project is to understand molecular pathways that regulate synapse formation in vivo using a vertebrate model with a focus on the underappreciated electrical synapse. Electrical synapses are sites of direct communication between neurons that allow the passage of ions and small molecules. They contribute extensively to neural circuit formation and function, both during development as well in adulthood where they contribute to sensory perception, interneuron processing, and motor output. However, the molecular mechanisms controlling the formation of electrical synapse are poorly understood. This proposal utilizes the zebrafish Mauthner circuit to investigate the genetics, cell biology, and biochemistry of electrical synapse formation and function. Mauthner neurons are individually identifiable and their pre- and postsynaptic partners, synapses, and function are exquisitely visualized in a living, vertebrate embryo. Classic forward and novel reverse genetic screens have identified the Connexins that form the inter-neuronal channels of the Mauthner electrical synapses, found that there are dedicated pre- and postsynaptic Connexins, and identified Neurobeachin, a post-Golgi trafficking protein, and Tight Junction Protein 1b (Tjp1b), a membrane-associated guanylate kinase (MAGUK) family scaffold, as being required for electrical synapse formation. These findings suggest that electrical synapses are comprised of a molecular complexity that is not generally appreciated; they further suggests that intricate biochemical mechanisms are required to control the formation, function, and plasticity of these critical sites of neuronal communication. Aim1 of this proposal examines the cell biological mechanisms of electrical synapse formation, examining the hypothesis that electrical synapses require the postsynaptic localization and function of Tjp1b to stabilize Connexins at the synapse. Aim2 examines the biochemical mechanisms of synaptogenesis, examining the hypothesis that a direct interaction between Tjp1b and the Connexins is required for localization to the synapse. Aim3 looks to expand the molecular repertoire of proteins required for electrical synapse formation, and provides a new view of electrical synapses as complex multi-molecular machines. Given that electrical synapses are essential to early developmental wiring of the brain, they may be intricately linked to developmental disorders of wiring. Indeed, both Neurobeachin and the MAGUKs are associated with autism and other neurodevelopmental disorders. The proposed studies will provide novel insight into the mechanisms of electrical synapse formation and provide a foundation for the identification of targets for therapy of complex neurodevelopmental disorders.